Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

Regulation of the production of immune interferon and cytotoxic T lymphocytes by interleukin 2

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

A requirement for nonspecific T cell factors in antibody responses to "T cell independent" antigens

Using murine splenic B cell preparations depleted of macrophages and rigorously depleted of T cells, we studied the role of nonspecific helper factors in in vitro antibody responses to T cell-independent (TI) type 1 and type 2 antigens. TNP-lipopolysaccharide, TNP-Brucella abortus, and DNP-liposomes containing lipid A were chosen as examples of TI type 1 antigens. DNP-Ficoll and DNP-liposomes without lipid A were chosen as TI type 2 antigens. Only the type 1 antigens were able to elicit significant, albeit very weak, responses without added helper factors. Both type 1 and 2 antigens required factors present in supernatants from concanavalin A-stimulated spleen cells (Con A SN) to stimulate optimum antibody responses. Interleukin 2- (IL 2) containing supernatant from the T cell hybridoma FS6-14.13 supported suboptimal responses to varying degrees with each TI antigen, in contrast to its lack of effect on responses to sheep red blood cells in the absence of additional factors. This activity of the FS6-14.13 supernatant was removed by absorption with the IL 2-dependent T cell line HT-2, suggesting that IL 2 was the active component. Another factor, IL-X, which is distinct from both IL 1 and IL 2 and is also found in Con A SN, was required in addition to IL 2 to achieve optimal responses with both types of TI antigens. These results clearly establish a role for factors derived from T cells in the activation of B cells by both type 1 and type 2 TI antigens.     

Enhancing effect of IFN-gamma on helper T cell activity and IL 2 production

A single injection of young murine immune interferon (IFN-gamma) in young (3 mo) or old (14 to 24 mo) mice 3 days before carrier-priming significantly enhances helper T cell activity of their spleen cells. Maximal enhancement is attained when IFN-gamma is injected once immediately before priming or for 4 consecutive days from the time of priming. Helper activity for anti-TNP antibody response was titrated in vitro by adding graded numbers of spleen cells from HRBC-primed mice of a given age to cultures containing a constant number of spleen cells from 3-mo-old normal mice and TNP-HRBC. When T cell-enriched spleen cells from HRBC-primed young or old mice, uninjected or injected with IFN-gamma, were separated by nylon wool filtration into passed (Thi) and adherent (Th2) cells, the helper activity of both T cell subpopulations was found to be enhanced by IFN-gamma injection. Helper activity of purified Th1 and Th2 cells was also increased by their in vitro preincubation with IFN-gamma. Furthermore, interleukin 2 (IL 2) production by mitogen-activated spleen cells from young and old mice is enhanced by addition of IFN-gamma to cultures. These data altogether indicate that IFN-gamma plays an important role in immunoregulation of helper T cell activity.     

Cloned T cells with "co-helper" activity. I. Biologic activities of the clone

Clones of sheep erythrocyte-(SRBC) specific helper T cells with the surface phenotype Thy-1+, Ly-1+, Ly-2- have been derived that grow in vitro in the absence of exogenous antigen or added growth factors. The IL 2-independent clone, 101.6 has been shown to produce a supernatant factor that augments the primary anti-SRBC but not anti-burro RBC responses of whole spleen cells or Ly-1 T plus B cell cultures. The supernatant does not help B cells directly. This augmenting activity is terminated "co-helper" because the enhancement requires the presence of normal Ly-1 T cells. The supernatant of 101.6 was not shown to contain IL 2; co-helper activity was distinguishable from IL 2 activity by absorption with SRBC but not with Con A blasts, and we observed that co-helper activity does not act on spleen cells that differ at the major histocompatibility complex.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Coordinate production by a T cell hybridoma of gamma interferon and three other lymphokine activities: multiple activities of a single lymphokine?

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.     

Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Constitutive production of a factor supporting B lymphocyte differentiation by a T cell hybridoma

To investigate the role of soluble T cell products during B cell differentiation more fully, we have produced T cell hybridomas by the fusion of normal helper T cells with the T cell lymphoma BW5147. In this report we describe the production of one such hybrid, 14G3, the subclone 14G3.1F2, and the functional activity of the constitutive product. The hybrid supernatant acts exclusively in antigen-nonspecific, but antigen-dependent, promotion of B cell differentiation. It is optimally effective in the presence of small amounts of EL4 supernatant. It does not itself contain any detectable IL 2 or BCGF or interferon activity, however. 14G3.1F2 activity is probably an important component of the conventional TRF preparations produced by mixed lymphocyte populations, and will be useful in further dissection of the contributions of different soluble T cell products to B cell differentiation.     

Retrovirus-mediated immunosuppression. II. FeLV-UV alters in vitro murine T lymphocyte behavior by reversibly impairing lymphokine secretion

Murine splenocytes and cloned murine T cells were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV) on lymphokine secretion. FeLV-UV can significantly depress the accumulation of IL 2 in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive helper T cell clone B6D/2-2m plus Con A. Inhibition of lymphokine accumulation in these cultures could not be attributed to absorption or inactivation of IL 2 by the FeLV-UV or to the FeLV-UV-induced production of substances which interfere with the IL 2 bioassay. Thus, FeLV-UV appears to block production and/or secretion of IL 2 by a direct inhibitory effect on IL 2-secreting murine T lymphocytes. Additional studies indicate that FeLV-UV impairs IL 2 production only if added very soon after lymphocyte contact with lymphokine-inducing agents and that IL 2 secretion resumes when FeLV-UV is removed from the culture. FeLV-UV also impairs accumulation of MAF (interferon-gamma?) in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive cytotoxic T lymphocyte clone B6D/2-7c plus Con A. The latter observation again suggests that FeLV-UV impairs lymphokine secretion by a direct effect on lymphokine-producing T lymphocytes. Furthermore, it suggests that FeLV-UV does not selectively impair production of IL 2 nor does it have selective inhibitory effects on helper T cells. Rather, FeLV-UV appears to have a general inhibitory effect on lymphokine production by T lymphocytes. Finally, concentrations of FeLV-UV which suppress MAF production by the CTL clone have little influence on the cytolysis mediated by the same cloned T cell population. Thus, the immunosuppressive influence of FeLV-UV is selective for phenomena associated with induction of new T lymphocyte functions, such as lymphokine secretion, and spares other immune functions already expressed by the same cells.     

Insulin-specific suppressor T cell factors

Murine antibody responses to heterologous insulins are controlled by MHC-linked immune response genes. Although nonresponder mice fail to make antibody when injected with nonimmunogenic variants of insulin, we have recently shown that nonimmunogenic variants stimulate radioresistant, Lyt- 1+2- helper T cells that support secondary antibody responses. However, the helper activity can not be detected unless dominant, radiosensitive Lyt-1-2+, I-J+ suppressor T cells are removed. In this paper we report that extracts of primed Lyt-2+ suppressor T cells contain insulin-specific suppressor factors (TsF) that are capable of replacing the activity of suppressor T cells in vitro. The activity of these factors is restricted by MHC-linked genes that map to the I-J region, and immunoadsorption studies indicated that they bind antigen and bear I-J-encoded determinants. Insulin-specific TsF consists of at least two chains, one-bearing I-J and the other the antigen-binding site. Furthermore, mixing of isolated chains from different strains of mice indicates that the antigenic specificity is determined by the antigen-binding chain and the MHC restriction by the H-2 haplotype of the source of the non-antigen-binding, I-J+ chain. Moreover, mixtures containing antigen-binding chain from allogeneic cell donors and I-J+ chain from responder cell donors have activity in cultures containing responder lymphocytes. This suggests that preferential activation of suppressor T cells, rather than differential sensitivity to suppression, results in the nonresponder phenotype to insulin.     

The interleukin 2 secretion defect in vitro in systemic lupus erythematosus is reversible in rested cultured T cells

Interleukin 2 (IL 2) secretion in response to mitogenic stimulation in vitro is strongly reduced in circulating T lymphocytes from patients with SLE. It is still not clear how this abnormality relates to the B cell hyperactivity in the disease. Some investigators proposed that an intrinsic T helper cell defect could lead to suppressor cell dysfunction and autoimmunity. Others have found that in fact increased suppressor cell activity can cause IL 2 hyposecretion. In the present study we report that the IL 2 secretion in response to PHA plus PMA by T cells from patients with SLE, which initially was decreased by a factor of 10 as compared with the IL 2 secretion in blood donor T cells, was restored when the T cells were rested for 2 to 3 days in culture before stimulation. IL 2 hyposecretion in SLE T cells and the kinetics of normalization in culture were not changed by the addition of normal adherent cells during the stimulation with PHA/PMA, occurred in the absence of significant cell death or proliferation or change of the T4:T8 cell ratio during the resting culture, were not due to a maturation of immature T6-positive cells (less than 1.5% T6 cells in SLE T cells), and also occurred in T8-depleted T4 cells alone. Furthermore, a normalization of IL 2 secretion took place in the presence of either SLE serum or normal serum, and the addition of fresh autologous T cells to 3-day-cultured SLE T cells did not cause suppression of IL 2 secretion. These data show that some rapidly reversible defect occurs in circulating T helper cells in SLE. That this could reflect an exhaustion of T helper cells that have been activated in vivo is discussed.     

Induction of CTL responses to alloantigens by a Db-specific T helper clone

A T cell helper clone was derived 2 yr ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in interleukin 2 (IL 2)-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2d origin and was found to be Thy-1+ and Lyt-1-2-. Clone 9, as well as supernatant factor(s) derived from it, were able to enhance the primary cytotoxic responses of Db responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hr of a 5-day culture period. When a low stimulator cell dose (10(4) cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions, clone 9 or its factor(s) could also synergize with IL 2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL 2-containing medium and clone 9 or its factor(s) was observed only when Db responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated Db spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of Db responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL 2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with Db cytotoxic T lymphocyte precursors (CTL-P).     

Functional diversity of T lymphocytes due to secretion of different cytokine patterns.

Different helper T cell subsets secrete different patterns of cytokines when stimulated by antigen. The TH1 and TH2 subsets differ in the secretion of at least eight cytokines, and three or more other cytokine secretion patterns also exist among both mouse and human T cell clones. Several properties of strong immune responses suggest that at least the TH1 and TH2 phenotypes can be present in vivo. As cytokines are major determinants of the functions of the T cells that produce them, these patterns lead to different properties of the T cell subsets. TH1 cells mediate several functions connected with cytotoxicity and local inflammatory reactions, and so these T cells are particularly effective at combating viruses and intracellular bacteria and parasites. TH2 cells are much more effective at stimulating B cells to produce antibody, and so should be more effective against free-living bacteria, and in inducing protective humoral immunity. Antibody and delayed inflammatory reactions are often mutually exclusive during immune responses, and this can be at least partially explained by cross-inhibition of TH1 and TH2 cells. A newly discovered cytokine, IL10, has been implicated as one of the cross-regulatory cytokines, as this TH2 product inhibits cytokine synthesis by TH1 cells.     

T3-T cell receptor (Ti) complex-independent activation of T cells by wheat germ agglutinin

The T3-Ti complex appears to play a central role in the activation of T cells by antigens and mitogens. Wheat germ agglutinin (WGA) is a unique lectin which inhibits T cell proliferation induced by mitogens, but it also induces marked IL 2 production by peripheral blood T cells. The pattern of responses induced by WGA suggests that this lectin may use a different mechanism of T cell activation other than the mechanism employed by the common T cell stimulants. We first investigated the production of IL 2 by Jurkat cells (E6-1) stimulated with WGA, before and after modulation of the surface T3-Ti complex. IL 2 production was markedly reduced after modulation of the T3 antigen from the cell surface when these cells were stimulated with PHA. In contrast, little change was observed in WGA-induced IL 2 production after modulation. Furthermore, we examined the effect of WGA on a T3-mutant of E6-1 cells (T3.1) which does not produce IL 2 in response to PHA or PHA plus PMA. WGA-stimulated T3.1 cells produced a significant amount of IL 2 with or without added PMA. In addition, a small but consistent rise in intracytoplasmic free calcium was observed when these cells were stimulated with WGA. These results demonstrate the presence of an alternative mechanism of T cell activation independent of the T3-Ti complex.     

T cell regulation of the immune response to infection in periodontal diseases

Although T cells have been implicated in the pathogenesis and are considered to be central both in progression and control of the chronic inflammatory periodontal diseases, the precise contribution of T cells to the regulation of tissue destruction has not been fully elucidated. Current dogma suggests that immunity to infection is controlled by distinct T helper 1 (Th1) and T helper 2 (Th2) subsets of T cells classified on the basis of their cytokine profile. Further, a subset of T cells with immunosuppressive function and cytokine profile distinct from Th1 or Th2 has been described and designated as regulatory T cells. Although these regulatory T cells have been considered to maintain self-tolerance resulting in the suppression of auto-immune responses, recent data suggest that these cells may also play a role in preventing infection-induced immunopathology. In this review, the role of functional and regulatory T cells in chronic inflammatory periodontal diseases will be summarized. This should not only provide an insight into the relationship between the immune response to periodontopathic bacteria and disease but should also highlight areas of development for potentially new therapeutic modalities.     

Characterization of a novel murine T cell-activating factor

Purified resting peripheral lymph node T cells can be activated to produce interleukin 2 (IL 2) and to proliferate in the presence of Concanavalin A (Con A) and an apparently novel lymphokine that we call T cell activating factor (TAF). TAF is biochemically distinct from IL 1, IL 2, IL 3, and other colony stimulating factors, IL 4 (BSF-1) and interferons. Furthermore, of the recombinant and natural cytokines tested, only IL 2 and TAF are active in the TAF assay. In the presence of Con A, TAF stimulates an increase in the steady-state level of IL 2 mRNA in T cells, the secretion of active IL 2 into the culture medium, and the proliferation of the T cells. We propose that TAF is a previously undescribed molecule the function of which is to stimulate IL 2 production by T cells that have encountered antigen, and we propose that TAF has an important role in primary T cell immune responses.     

Antigen-independent activation of memory cytotoxic T cells by interleukin 2

Culture supernatants from mitogen- or antigen-activated murine spleen cells are capable of causing reexpression of specific cytolytic activity from inactive memory cytotoxic T lymphocytes (CTL) in the absence of the original priming antigen. We have demonstrated that memory CTL from cytolytically inactive day 14 MLC cells are induced to reexpress high levels of specific cytotoxic activity after incubation with IL 2. Highly purified IL 2 was shown to induce levels of lytic activity comparable with that induced by supernatants from secondary mixed lymphocyte cultures (secondary MLC SN), suggesting that only IL 2 is necessary for the reactivation process. Moreover, only Lyt-2+ cells are necessary for reactivation inasmuch as inactive MLC cells depleted of Lyt-1+ cells by treatment with antibody and complement, followed by FACS selection of Lyt-2+ cells, were efficiently reactivated by IL 2. Because IL 2 is considered a proliferative signal, we examined whether proliferation was requisite for reactivation of memory CTL by IL 2. In the presence of cytosine arabinoside, which effectively inhibited proliferation, IL 2 was capable of reactivating memory CTL as efficiently as antigen, thus implying a differentiative role for IL 2 in secondary CTL activation. Reactivation of CTL by IL 2 and antigen appear to be functionally distinct events, because antigen but not IL 2 could trigger immune interferon release, although either IL 2 or antigen induced high levels of cytotoxicity. We propose that resting, memory CTL retain a heightened level of expression of IL 2 receptors as compared with naive CTL precursors, and thus are able to respond directly to exogenous IL 2. The consequences of this are proliferation and reexpression of specific killing activity, but this signal is not sufficient to induce immune interferon secretion. Rather, it appears that a signal via the antigen receptor is necessary for release of this lymphokine.